PROJECT SUMMARY Telomerase maintains telomere length and prevents cellular senescence. It is upregulated in 90% of cancers, suppressed in most non-cancer tissues, and is a primary driver of tumor cell immortality. No clinical agent acting on telomerase has been approved for human use. A hurdle for approaches that directly inhibit the action of telomerase is that therapeutic effect is observed only after many population doublings?a process that can take months, even for rapidly dividing cells. G-quadruplexes (G4s) and i-motifs (iMs) serve as master switches to control gene expression. Human telomerase reverse transcriptase (hTERT, the catalytic portion of the telomerase holoenzyme) transcription can be attenuated through the enhanced folding of the tandem G4s in the promoter region. A screen of the NCI diversity set resulted in identification of a family of drug-like small molecules that enhance the kinetic folding rate of the G4 silencer element. In vitro studies support this singular mechanism of action, resulting in reduced telomerase production and telomere shortening. Significantly, tumor cells treated with the identified molecules enter apoptosis in days, suggesting a unique downstream effect. The rapid onset of hTERT down-regulators stands in contrast to the delayed action of classic telomerase inhibition. The explanation is that hTERT down-regulation kills cancer cells primarily through mitochondrial effects, not downstream telomerase deactivation. In the absence of hTERT down-regulation, nuclear telomerase is shuttled to the mitochondria. There it protects mitochondrial function against apoptosis by up- regulation of the anti-apoptotic peptide BCL2 and elevation of reactive oxygen species (ROS) scavengers. hTERT down-regulation attenuates BCL2 and ROS scavenger production, resulting in rapid apoptosis. Prostate cancer (PCa) is the most common cancer in men and the number two killer. For most patients, the last line of defense is docetaxel (Taxotere). However, response rates approach 0% within 3 years. hTERT mRNA in the serum or plasma affords greater diagnostic and prognostic accuracy for PCa than the widely used prostate-specific antigen assay. Human-derived wild-type and Taxotere-resistant (TxR) PCa cells are sensitive to hTERT down-regulators and enter apoptosis within days. The forward-looking part of this proposal is the translation of the hTERT down-regulation compound series onto a commercialization path for the treatment of TxR PCa. The project plan is built on a multiparametric, data-driven hit-to-lead approach to identify molecules with optimized in vitro efficacy, safety, and drug-like profiles. The culmination of Phase I of this proposal is an in vivo experiment in a validated mouse model of PCa. Success will enable a lead optimization campaign with an objective of clinical candidate identification.